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PreprintCited by 2Wnt/β-catenin signaling in development and cancerLiver physiology and pathologyCancer-related Molecular Pathways

Hepatocyte Cell Cycle Progression Depends on a Transcriptional Repressor Cascade Downstream of Wnt Signaling

Open access
Jin, Yinhua, Anbarchian, Teni, Wu, Peng, Sarkar, Abby, Fish, Matt, Nusse, Roel·2021·bioRxiv (Cold Spring Harbor Laboratory)
DOI 10.1101/2021.10.15.464616
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Abstract

Cell proliferation is tightly controlled by inhibitors that block cell cycle progression until growth signals relieve this inhibition. In several tissues including the liver, transcriptional repressors such as E2F7 and E2F8 function as inhibitors of mitosis and promote polyploidy, but how growth factors release these mitotic inhibitors to facilitate cell cycle progression is unknown. We describe here a newly identified mechanism of cell division control in which Wnt/βcatenin signaling in the postnatal liver maintains active hepatocyte proliferation through Tbx3 , a Wnt target gene. TBX3 directly represses transcription of E2f7 and E2f8 , promoting a low ploidy state and cell cycle progression. This sequential transcriptional repressor cascade, initiated by Wnts, provides a new paradigm for exploring how a commonly active developmental signal impacts cell cycle completion.

AI snapshot

Written from the paper’s full text.

TL;DR

In postnatal mouse liver, Wnt/β-catenin signaling maintains hepatocyte proliferation by inducing Tbx3, which directly represses the mitotic inhibitors E2f7 and E2f8. This transcriptional repressor cascade promotes a low-ploidy, dividing hepatocyte state.

Key findings

  • Tbx3 acts downstream of Wnt signaling and promotes hepatocyte proliferation: Tbx3 knockdown slowed cultured hepatocyte proliferation and increased polyploid cells, while Tbx3 overexpression increased proliferation even without CHIR-activated Wnt signaling and increased the percentage of diploid cells.
  • Tbx3 is required in vivo to prevent hepatocyte polyploidization: Axin2-rtTA-driven Tbx3 KO livers showed a significant decrease in diploid nuclei (2N: 39.3% in Control vs 7.57% in Tbx3 KO), and Alb-CreERT2-driven Tbx3 KO livers had larger nuclei and increased ploidy without changes in liver shape or relative mass.
  • TBX3 directly represses E2f7 and E2f8: ChIP-seq and ChIP-qPCR showed TBX3 binding to the E2f7 promoter and E2f8 enhancer; E2f7/E2f8 transcripts increased in Tbx3 KO/KD and decreased with Tbx3 overexpression; luciferase reporters containing E2f7 or E2f8 regulatory regions were repressed in HepG2 cells.
  • Genetic epistasis showed that deleting E2f7 and E2f8 along with Tbx3 rescues the polyploid phenotype: Tbx3-E2f7-E2f8 triple knockout GFP+ nuclei were indistinguishable from controls in ploidy and size. Human ENCODE ChIP-seq also showed TBX3 binding to E2F7 and E2F8 enhancers.

Methods

  • Mouse genetics: conditional Tbx3 loss using Axin2-rtTA; TetO-H2B-GFP; TetO-Cre; Tbx3 f/f with doxycycline from postnatal week 2 to week 4, and Alb-CreERT2; Tbx3 f/f with tamoxifen at postnatal day 3; also Wls KO and CRISPR/Cas9 Apc deletion models. Ploidy was measured by propidium iodide flow cytometry of hepatocyte nuclei/cells, and nuclear area by immunofluorescence.
  • Primary hepatocyte culture: hepatocytes isolated from 8-week-old mice by two-step collagenase perfusion, expanded in Wnt-dependent media, and manipulated by Tbx3 shRNA knockdown or Sleeping Beauty-mediated Tbx3-HA overexpression; proliferation was assessed by growth curves and ploidy by flow cytometry.
  • Molecular assays: ChIP-seq and ChIP-qPCR for TBX3 binding, qRT-PCR and mRNA in situ hybridization for Tbx3/E2f7/E2f8 expression, and luciferase reporter assays in HepG2 cells using E2f7 promoter or E2f8 enhancer regions.

Limitations

  • The paper states that whether Wnt signaling likewise regulates mitosis by suppressing E2f7 and E2f8 in other tissues and contexts remains to be determined.
  • Evident from the text: functional evidence is largely from mouse in vivo models and mouse primary hepatocyte culture; human data are limited to TBX3 expression in pericentral hepatocytes and ENCODE ChIP-seq binding, without human functional loss- or gain-of-function experiments.
  • Evident from the text: ploidy and nuclear area are used as the main readouts for M-phase completion, rather than direct quantification of mitotic progression markers.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1101/2021.10.15.464616

Cites

51 of 52 references verified0 citation errors · 1 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

21 of 31 cited sources checked · 8 supported · 0 flagged · accuracy not yet measured

  • Findingown data

    Tbx3 is a target of Wnt signaling in the liver.

    “We verified that Tbx3 is a target of Wnt signaling in the liver, by either over-activating or eliminating the Wnt pathway in the tissue (Figure .1A, arrows point to TBX3 + Apc KO cells, arrowheads point to TBX3 -Wls KO cells), in agreement with data from other contexts”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Aydoğdu et al. 2018 source

      The title in our copy identifies TBX3 as acting downstream of canonical WNT signaling in mouse ureteric mesenchyme, supporting the citing sentence’s reference to data from another context. It does not establish that TBX3 is a WNT target in the liver; the liver experiments are presented as the citing authors’ own work. (LLM, conf=0.79)

      Experimental — accuracy not yet measured
    • cites Eblaghie et al. 2004 source
    • cites Renard et al. 2007 source
  • Premisecitation

    E2F7 and E2F8 are responsible for cell cycle arrest in the liver.

    “Two members of the E2F family of cell cycle regulators, E2F7 and E2F8, are responsible for this form of cell cycle arrest in the liver (Chen et al. 2012;Pandit et al. 2012).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Chen et al. 2012 source

      The Results sections on hepatocyte endocycles and endocycle-related genes show that E2F7 and E2F8 help suppress mitosis in mouse hepatocytes, with E2F8 identified as the principal regulator. The paper describes a mitotic block that permits continued DNA replication, not a general arrest of the cell cycle; the claim omits that distinction. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites Pandit et al. 2012 source
  • Premisecitation

    Deleting E2f7 and E2f8 from liver cells results in completion of M phase and generation of diploid daughter cells.

    “Deleting E2f7 and E2f8 from these cells results in completion of M phase and the generation of diploid daughter cells (Chen et al. 2012;Pandit et al. 2012).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Chen et al. 2012 source
    • cites Pandit et al. 2012 source
  • Premisecitation

    Tbx3 is expressed in a zonated pattern during postnatal liver growth.

    “The transcriptional repressor Tbx3, a gene required for embryonic liver development (Khan et al. 2020;Ludtke et al. 2009;Suzuki et al. 2008), is also expressed in a zonated pattern during postnatal liver growth (fig.S1A) (Wang et al. 2015;Zhao et al. 2019).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Wang et al. 2015 source

      The “Axin2+ cells self-renew” section reports that Tbx3 expression is confined to a single layer of pericentral cells, supporting zonated expression. The Methods identify the mice used for the Tbx3 in situ images as eight-week-old animals; the paper studies adult liver homeostasis, not expression during postnatal liver growth specifically. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites Zhao et al. 2019 source

      The Results section ('Injury induces de novo Wnt response in mid-lobular hepatocytes'; page unavailable) describes a zonated, pericentral Tbx3 expression pattern in the uninjured adult liver and its return after injury. It does not establish that this pattern occurs during postnatal liver growth, the stage specified in the claim. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • Premisecitation

    In the liver, DNA synthesis is uncoupled from cell proliferation due to the activity of E2F7 and E2F8.

    “Therefore, in the liver, DNA synthesis is uncoupled from cell proliferation due to the activity of E2F7 and E2F8.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Chen et al. 2012 source
    • cites Pandit et al. 2012 source
  • Classified as probably not a substantial claim. 10
  • May combine two claims. 10
  • The quote only partly states this claim. 1

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    These mitotic inhibitors are expressed in several other tissues besides the liver where they are also implicated in arresting the cell cycle.

    “These mitotic inhibitors are expressed in several other tissues besides the liver where they are also implicated in arresting the cell cycle (Matondo et al. 2018;Mizuno et al. 2019;Pandit et al. 2012;Qi et al. 2015).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Matondo et al. 2018 source

      The Results (§3.1; page unavailable) detect E2f7/8 transcripts in multiple adult tissues, and the Discussion (§4; page unavailable) says these factors repress genes important for cell-cycle progression in pancreatic, liver, and placental cells. The paper links them to abortive cell cycles and polyploidization, but does not establish that they arrest the cell cycle in those tissues generally. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites Mizuno et al. 2019

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Pandit et al. 2012 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    High Wnt/βcatenin signaling activity during cell division suggests a link between Wnt signaling and mitosis.

    “High Wnt/βcatenin signaling activity during cell division has suggested a link between Wnt signaling and mitosis through unknown mechanisms (Benham-Pyle et al. 2016;Niehrs and Acebron 2012).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Benham-Pyle et al. 2016 source
    • cites Niehrs and Acebron 2012 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Activating mutations in Wnt signaling components are common in liver cancer.

    “Interestingly, activating mutations in Wnt signaling components are common in liver cancer (Nusse and Clevers 2017;Wang et al. 2019)”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Nusse and Clevers 2017 source
    • cites Wang et al. 2019 source

      Section 4.1 reports activating CTNNB1 mutations in 15–25% of hepatocellular carcinomas, and Section 4.3 reports them in 60–80% of hepatoblastomas. The unqualified claim is broader than those findings: Section 4.2 says mutations in related genes are less frequent in cholangiocarcinoma. Page numbers were not provided in our copy. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    In several tissues including the liver, transcriptional repressors E2F7 and E2F8 function as inhibitors of mitosis and promote polyploidy.

    “In several tissues including the liver, transcriptional repressors such as E2F7 and E2F8 function as inhibitors of mitosis and promote polyploidy”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Chen et al. 2012 source
    • cites Pandit et al. 2012 source

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    The postnatal liver expands rapidly in a Wnt/βcatenin signaling-dependent way and switches from initially containing diploid hepatocytes to becoming mostly polyploid.

    “The postnatal liver expands rapidly in a Wnt/βcatenin signaling-dependent way and switches from initially containing diploid hepatocytes to becoming mostly polyploid (Apte et al. 2007;Monga et al. 2001).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Apte et al. 2007 source
    • cites Monga et al. 2001 source

      The Discussion describes changes in Wnt/β-catenin signaling during liver regrowth after partial hepatectomy in adult rats and suggests that β-catenin may help initiate cell proliferation. It does not establish that postnatal liver expansion depends on this pathway or describe a shift from diploid to mostly polyploid hepatocytes (searched: postnatal, diploid, polyploid). (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Tbx3 is a gene required for embryonic liver development.

    “The transcriptional repressor Tbx3, a gene required for embryonic liver development (Khan et al. 2020;Ludtke et al. 2009;Suzuki et al. 2008)”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites Khan et al. 2020 source

      The developmental overview says Tbx3 is expressed in the developing mouse liver, and the cancer section reports that it increases proliferation of hepatic progenitor cells. The References section lists studies finding that Tbx3 promotes liver-bud expansion and controls hepatic progenitor-cell fate, but the review passages do not establish the stronger claim that Tbx3 is required for embryonic liver development. No page numbers are available in our copy. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites Ludtke et al. 2009 source
    • cites Suzuki et al. 2008 source

  • May combine two claims.

    Premisecitation

    In mouse and human liver, Wnt ligands are produced by endothelial cells of central vein branches and nearby sinusoids, creating a zonated expression pattern of Wnt target genes and key metabolic enzymes in pericentral hepatocytes.

    “In the mouse and human liver, Wnt ligands are produced by endothelial cells of the central vein branches and the nearby sinusoids (McEnerney et al. 2017;Wang et al. 2015;Zhao et al. 2019), creating a zonated expression pattern of Wnt target genes and key metabolic enzymes in pericentral hepatocytes (Halpern et al. 2017).”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites McEnerney et al. 2017 source

      The Discussion (page unavailable) reports rodent evidence that central-vein endothelial cells supply Wnt ligands to adjacent zone-3 hepatocytes. It says endothelial-cell collection is still needed to validate this mechanism in human liver, and it does not establish Wnt production by endothelial cells of nearby sinusoids. The later claim about zonated target genes has a different citation in the citing sentence. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites Wang et al. 2015 source

      The “Central vein endothelium acts as a Wnt-producing niche” section reports Wnt2 and Wnt9b production by central-vein endothelial cells, and the Discussion specifies that Wnt2 is also expressed in sinusoidal endothelial cells. The experiments described are in mice; these passages do not establish the claim for human liver. The later zonation clause is attributed to a different citation, not to this paper. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Zhao et al. 2019 source

      The Results section (‘Upregulation of endogenous and exogenous Wnt ligands following injury’; page unavailable) says that Wnt2 and Wnt9b are expressed by central-vein endothelial cells in uninjured liver, and Wnt2 by sinusoidal endothelial cells. The paper’s observations concern mouse liver; our copy does not establish the same endothelial Wnt production in human liver. The subsequent zonated-expression clause is attributed to Halpern et al., not Zhao et al. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    E2f7 and E2f8 as well as polyploidy are known to suppress tumorigenesis.

    “while E2f7 and E2f8 as well as polyploidy are known to suppress tumorigenesis (Kent et al. 2016;Moreno et al. 2021;Wilkinson et al. 2019;Zhang et al. 2018).”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites Kent et al. 2016 source

      The “Tumor suppressive function of E2F8” section reports that E2f8 deletion promoted liver cancer in mice and that combined loss of E2f7 and E2f8 led to spontaneous liver cancer. The paper also says E2F8 is critical for hepatocyte polyploidy, but it does not establish that polyploidy itself suppresses tumorigenesis. Its tumor-suppression finding is specific to liver cancer in mice, rather than tumorigenesis generally. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites Moreno et al. 2021 source

      The Results section reports that E2f7 and E2f8 overexpression inhibited tumorigenesis in mouse liver cancers. Our copy does not establish the separate claim that polyploidy suppresses tumorigenesis; its reference to polyploidization concerns E2F8’s role in that process, not a tumor-suppressing effect. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites Wilkinson et al. 2019 source

  • May combine two claims.

    Findingown data

    CHIR alone enhances proliferation of hepatocytes, but not to the same degree as Tbx3 overexpression.

    “As expected, CHIR alone enhanced proliferation of hepatocytes albeit not to the same degree as Tbx3 overexpression (Figure 1D).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Tbx3 knockdown with shRNA #334009 results in a minor increase in the percentage of polyploid cells compared to Control.

    “Tbx3 KD with shRNA #334009 resulted in a minor increase in the percentage of polyploid cells, compared to Control in Fig. 1C.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Mean nuclear area is not significantly different between Control and Tbx3-E2f7-E2f8 TKO hepatocytes (P=0.2249).

    “P=0.2249, t-test with Welch's correction comparison of mean nuclear area from biological replicates.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    AlbCreERT2-driven loss of Tbx3 does not impact liver size or morphology.

    “AlbCreERT2-driven loss of Tbx3 does not impact liver size or morphology.”
    • Also stated in: § Additional information · plain
      “Photographs of Control (Tbx3 f/f ) and Tbx3 KO (Alb-CreERT2; Tbx3 f/f ) livers at postnatal weeks 2 and 12 show no changes to liver morphology.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The genetic interactions imply a linear pathway whereby Wnt activates Tbx3, which represses the mitotic inhibitors E2f7 and E2f8; this relieves cell cycle arrest and maintains hepatocytes in a diploid, dividing state.

    “These genetic interactions imply a linear pathway whereby Wnt activates Tbx3, which represses the mitotic inhibitors E2f7 and E2f8; this therefore relieves cell cycle arrest and maintains hepatocytes in a diploid, dividing state.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    In Tbx3 KO livers, cells are able to complete S phase but not M phase.

    “indicating that cells were able to complete S phase but not M phase.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    TBX3 promotes a low ploidy state and cell cycle progression.

    “TBX3 directly represses transcription of E2f7 and E2f8, promoting a low ploidy state and cell cycle progression.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    In the liver, M phase is regulated by Wnt signals through the Wnt target transcription factor Tbx3, which in turn represses the mitotic inhibitors E2f7 and E2f8.

    “In this work we have shown that in the liver, M phase is regulated by Wnt signals through the Wnt target transcription factor Tbx3, which in turn represses the mitotic inhibitors E2f7 and E2f8.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. The quote only partly states this claim.

    Study design and conductown data

    The ChIP-seq binding profiles for TBX3 at the E2f7 and E2f8 loci are representative of two independent biological replicates.

    “Profiles are representative of two independent biological replicates.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Tbx3 knockdown is efficient in hepatocytes.

    “Efficient knockdown (KD) of Tbx3 in hepatocytes.”
    Quote in the paperSubstantial claim
18 of 18

  • The paper relies on this without support.

    Tbx3 is a transcriptional repressor.

    “To identify the possible mechanisms of TBX3 function, a known repressor of transcription, we investigated potential target genes using Chromatin immunoprecipitation-sequencing (ChIP-Seq) in Tbx3-overexpressing mouse primary hepatocytes.”

  • The paper relies on this without support.

    CHIR99021 activates Wnt signaling in primary hepatocytes.

    “even in the absence of CHIR-activated Wnt signaling (Figure 1D)”
2 of 2

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • E2f7 luciferase insert is called both an enhancer and a promoter

    Minor

    The Results describe the E2f7 sequence added to the luciferase reporter as an enhancer, but the assay methods identify the E2f7 insert as a 122 bp promoter region.

1 of 1

Cited works that were retracted

Works this paper cites that were later retracted, from the retraction notices publishers deposit. Major when the notice came out before this paper was published.

  • Cites a retracted work: 10.3727/096504017x15145624664031

    Info

    The cited work was retracted on 2026-09-14 (notice 10.32604/or.2026.092122), not before this paper was published (2021-10-16).

    Cited work 10.3727/096504017x15145624664031 · retracted 2026-09-14 · notice 10.32604/or.2026.092122 · this paper published 2021-10-16

1 of 1

Cites

1 retracted

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Related

Authors
Jin, Y., Anbarchian, T., Wu, P., Sarkar, A., Fish, M., & Nusse, R.
Affiliations
Howard Hughes Medical Institute (US)
ORCID
Venue
bioRxiv (Cold Spring Harbor Laboratory)
Publisher
openRxiv
Published
2021-10-16
DOI
10.1101/2021.10.15.464616
OpenAlex
W3207784456
Cited by
2
References
51
Citations by year
2023: 1 · 2022: 1
Type
posted content
Language
en
Open access
Locations
bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
Funders
  • National Science Foundation (1147470)
  • U.S. Department of Defense (#W81XWH-17, W81XWH)
  • Howard Hughes Medical Institute
  • Damon Runyon Cancer Research Foundation (DRSG-28P-19)
  • DOD Peer Reviewed Cancer Research Program
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Wnt/β-catenin signaling in development and cancer
  • Health Sciences › Medicine › Hepatology › Liver physiology and pathology
  • Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
Keywords
hepatocyte proliferation, cell cycle progression, Wnt/β-catenin signaling, TBX3, E2F7, E2F8, transcriptional repression, polyploidy, liver development, cell division regulation

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